Molecular diagnostics are an increasingly important part of biotechnology. The ability to detect small quantities of genomic, proteomic, and other biological materials enables sensitive clinical tests to be performed, as well as enabling laboratory research that affects drug development and functional biology. Molecular sensing modalities include radioactivity, mass spectroscopy, and electrical and optical techniques. The worldwide market for molecular biomarkers, diagnostics, and related services exceeded $6 billion in 2003.
While current technology is quite useful, it is not as sensitive or as specific as is desired, and there is room for improvement in the speed and economics of the diagnostics. The utility of many current genomic assays is limited by the need to replicate sample material using techniques like Polymerase Chain Reaction (PCR), which can add noise, take time to process, and may add cost through royalty payments. Even with large material samples, the results of gene chip readers have significant statistical variation. Consequently, there is great interest in techniques that improve the ability to unambiguously detect specific molecules in very small quantities. Nanotechnology has been identified as one approach that can potentially improve the sensitivity of molecular diagnostics.
My new approach, which will hereinafter be discussed, is to use a novel family of nanostructured materials to develop a sensitive optical technique for molecular diagnostics that uses Surface Enhanced Raman Scattering (SERS) or other optical detection techniques. Raman spectroscopy is a non-invasive technique that requires little material preparation and can provide an essentially unique signature for biological and many other molecules. Nanometer-scaled conducting materials, through coupling to surface plasmon modes, can greatly enhance the Raman signal so that the diagnostic can be performed in a practical setting with minute amounts of material.
I will hereinafter discuss the use of a novel substrate material that satisfies all of the requirements for successful application of SERS to molecular diagnostics. Namely, the novel substrate material is inexpensive to produce, it can be precisely tailored for maximally enhancing the Raman signal, and it can easily be micropatterned for use as an array. As I detail below, the novel substrate can also be used to separate analytes that are in solution with the use of microfluidics and electrochemistry that are easily co-manufactured. The novel substrate has large effective surface area and can be tailored to enhance additional optical detection techniques such as fluorescence. This enables the material to serve as a platform for a variety of molecular diagnostics.
In vitro molecular diagnostics can be performed with a variety of sensing modalities that measure optical, electrical, radioactive, or mass spectroscopic properties of the material under test (i.e., the analyte). In most scenarios, the analyte is processed so it is selectively bonded to a compound in the apparatus. Sometimes either the material or a mating compound is tagged with a label such as a fluorophore, nanosphere, or another agent that is then detected to indicate the presence and/or concentration of the principal analyte or analytes.
The present invention is, among other things, concerned with a photonic diagnostic technique (i.e., Raman spectroscopy utilizing Surface Enhanced Raman Scattering, or SERS) that can be label-free and may or may not require the analyte to be bonded to another compound.
Raman scattering is the process whereby an optical photon inelastically scatters off a molecule by coupling with the vibrational modes of the molecule. The scattered photon energy is reduced (Stokes) or augmented (anti-Stokes) by the energy of the vibrational mode. The Raman scattered light has a detailed spectrum that is essentially unique for biological and many other molecules as it encodes all of the bonds present in the molecule, and may indicate the conformation of the molecule as well. Raman spectroscopy (RS) is the technique whereby the spectrum is measured by quantitatively recording the Raman scattered light as a function of wavelength or wavenumber (cm−1) when a monochromatic (e.g., a laser) beam illuminates the sample. An example of a Raman spectrum is shown in FIG. 1.
One barrier to the use of Raman spectroscopy is the small cross-section for Raman scattering, a factor of ˜1014 less than the cross-section for fluorescence. This problem is mitigated when the molecule is adsorbed onto, or is near, a conductive surface with structure at the appropriate nanometer-sized scale. Then, the incident electromagnetic field, e.g., the laser, the plasmon modes of the conduction electrons, and the molecular vibrational modes strongly couple and greatly enhance the Raman scattering cross-section. This electromagnetic (EM) enhancement can increase the cross-section by up to a factor of 1014, locally, and by a factor of 104-108 averaged over the ensemble of molecules nearby or adsorbed on a conducting surface. In addition to the EM enhancement, additional enhancement can come from chemical interactions or from a resonance of the molecule with the input laser (i.e., the Raman pump) wavelength. The latter effect is usually termed Surface Enhanced Resonance Raman Scattering (SERRS).
The basic correlation of nanoscale structure and surface plasmon excitation is apparent from Mie scattering theory. The characteristic structure size for effective SERS enhancement ranges from tens of nm for isolated metal particles to several hundred nm for nanostructured surfaces. The optimum feature size for maximum enhancement scales with the pump wavelength. Both theoretical and experimental studies have shown that the EM enhancement is maximized where particles are nearly or actually touching, or generally where the surface is discontinuous and electric fields are presumed to be large. There is evidence that periodic structures increase the SERS enhancement. However, it is generally acknowledged that detailed knowledge and prediction of the surface enhancement phenomenon is not completely understood at this time.
Many substrates have been used for SERS. Initial work used metal electrodes that were electrochemically etched to produce nanoscale roughness. Those substrates were particularly unpredictable and often changed their properties over time due to electrochemical reactions. Molecules in solution have been analyzed with SERS by introducing nano-sized metal particles into the solution. These particles have included silver and other metal colloids and, more recently, nano-sized spheres that are produced by a variety of means. Over the years, SERS substrates have generally been made by forming a nanostructure, then evaporating a metalized layer onto the nanostructure. The underlying substrate has included lithographically etched materials, chemically etched materials, and a self-assembled monolayer of plastic nanospheres. Additional techniques for forming SERS substrates involve evaporating metal films onto glass slides—this can include depositing metal islands on the glass slide by not uniformly covering the surface of the slide, and nanopatterning the surface of the slide by using the interstices of a self-assembled nanosphere layer as apertures in a technique called nanosphere lithography. Recently, a substrate has been announced that uses a metalized photonic crystal formed by semiconductor lithography. Another substrate with interesting properties, but which is probably not affordably manufacturable, is e-beam lithography of silicon.
The interest in SERS as an analytical technique comes from several features. The signal from SERS can be larger than the fluorescence signal due to the surface enhancement and the shorter lifetime of Raman excitation relative to fluorescence, which can enable more scattering events per molecule per unit time. The SERS spectrum is typically 10˜100 times narrower than the typical fluorescence spectrum. This addresses one serious issue with fluorescence studies, i.e., the number of different labels that can be distinguished in the same assay. With fluorescence labels, a maximum of 12 labels can be distinguished. This number can increase, significantly, if SERS-active labels are used. At least one company, Nanoplex Technologies, Inc. of Mountain View, Calif., is focused on developing labeling nanoparticles that are SERS-active.
Moreover, the SERS spectrum is essentially unique for each analyte. This offers the opportunity to identify specific analytes that do not need to be labeled with a fluorescent dye. This same feature can also enable label-free binding detection. Many assays are designed to measure the binding or conjugation of two complementary materials—e.g., a protein-ligand, a DNA strand-oligonucleotide, etc. The SERS spectrum has been shown by to exhibit variation that indicates when binding has occurred, as is seen in FIG. 2. SERS has been used to quantitatively detect the amount of an analyte and measure time-varying signals indicative of binding kinetics, as is seen in FIG. 3. For the case of a protein (avidin) and a small molecule (biotin), a Raman spectral signature of the bound complex shows variation in the protein spectrum (tryptophan bands) and the presence of a biotin peak at 690 cm−1 which has information about the structure and function of the bound complex, as is seen in FIG. 4.
With the surface enhancement in SERS analyses, very small quantities of material can be detected using spatially-resolved detection. Some experiments have achieved attomole sensitivity and, for isolated particles, detection of single molecules has been achieved. There have been numerous papers on SERS and related studies of plasmon resonance with nanoscaled structures. There have been hundreds of papers per year on this topic since 1995.
As many of the advantages of SERS have been known for years, and with the level of interest in the literature, it is instructive to understand why SERS analysis has not become more prevalent. One major reason often cited in the literature is the lack of availability of a suitable substrate material. Many substrates exhibit a wide variation in sensitivity from realization to realization, or over time. Other substrates are expensive or difficult to prepare. The development of quantitative analysis using SERS depends on the availability of substrates that are inexpensive (i.e., easy) to produce, are readily reproducible, and offer the potential to tailor the surface features for strong Raman scattering enhancement. While some of the SERS substrates I have described above meet one or more of these criteria, no substrate is currently commercially available that meets all of them.